- Stereochemical Evidence for Monomeric Thiometaphosphate as an Intermediate in the Hydrolysis of (Rp) and (Sp)-Deoxyadenosine 5'--β-thiodiphosphate
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(Rp)-Deoxyadenosine 5'--β-thiodiphosphate is hydrolysed in (18O)water at pH 7.2 (where it is present as its trianion) to AMP and inorganic thiophosphate with complete inversion of configuration at phosphorus, whereas at pH 4.5 (where it is present as its dianion) hydrolysis occurs with 50percent racemisation and 50percent inversion, indicating that 'liberated' metathiophosphate can exist and has a finite lifetime in aqueous solution under these conditions.
- Harnett, Sara P.,Lowe, Gordon
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Read Online
- Synthesis of Terminal Ribose Analogues of Adenosine 5′-Diphosphate Ribose as Probes for the Transient Receptor Potential Cation Channel TRPM2
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TRPM2 (transient receptor potential cation channel, subfamily M, member 2) is a nonselective cation channel involved in the response to oxidative stress and in inflammation. Its role in autoimmune and neurodegenerative diseases makes it an attractive pharmacological target. Binding of the nucleotide adenosine 5′-diphosphate ribose (ADPR) to the cytosolic NUDT9 homology (NUDT9H) domain activates the channel. A detailed understanding of how ADPR interacts with the TRPM2 ligand binding domain is lacking, hampering the rational design of modulators, but the terminal ribose of ADPR is known to be essential for activation. To study its role in more detail, we designed synthetic routes to novel analogues of ADPR and 2′-deoxy-ADPR that were modified only by removal of a single hydroxyl group from the terminal ribose. The ADPR analogues were obtained by coupling nucleoside phosphorimidazolides to deoxysugar phosphates. The corresponding C2″-based analogues proved to be unstable. The C1″- and C3″-ADPR analogues were evaluated electrophysiologically by patch-clamp in TRPM2-expressing HEK293 cells. In addition, a compound with all hydroxyl groups of the terminal ribose blocked as its 1″-β-O-methyl-2″,3″-O-isopropylidene derivative was evaluated. Removal of either C1″ or C3″ hydroxyl groups from ADPR resulted in loss of agonist activity. Both these modifications and blocking all three hydroxyl groups resulted in TRPM2 antagonists. Our results demonstrate the critical role of these hydroxyl groups in channel activation.
- Baszczyňski, Ond?ej,Watt, Joanna M.,Rozewitz, Monika D.,Guse, Andreas H.,Fliegert, Ralf,Potter, Barry V.L.
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p. 6143 - 6157
(2019/05/24)
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- Catalysts for reversing formaldehyde adducts and crosslinks
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Catalysts act to release formaldehyde cross-linking that occurs in biological samples. Thus, contacting catalysts to formaldehyde fixed samples is a useful way to render biological components of the samples, including nucleic acids or proteins, more accessible to detection and characterization.
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Paragraph 0073
(2019/01/10)
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- Fully automated continuous meso-flow synthesis of 5′-nucleotides and deoxynucleotides
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The first continuous meso-flow synthesis of natural and non-natural 5′-nucleotides and deoxynucleotides is described, representing a significant advance over the corresponding in-flask method. By means of this meso-flow technique, a synthesis with time consumption and high-energy consumption becomes facile to generate products with great efficiency. An abbreviated duration, satisfactory output, and mild reaction conditions are expected to be realized under the present procedure.
- Zhu, Chenjie,Tang, Chenglun,Cao, Zhi,He, Wei,Chen, Yong,Chen, Xiaochun,Guo, Kai,Ying, Hanjie
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p. 1575 - 1581
(2015/02/19)
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- Immobilized Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) as a high performing biocatalyst for the synthesis of purine arabinonucleotides
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Fruit fly (Drosophila melanogaster) deoxyribonucleoside kinase (DmdNK; EC: 2.7.1.145) was characterized for its substrate specificity towards natural and non-natural nucleosides, confirming its potential in the enzymatic synthesis of modified nucleotides. DmdNK was adsorbed on a solid ion exchange support (bearing primary amino groups) achieving an expressed activity >98%. Upon cross-linking with aldehyde dextran, expressed activity was 30-40%. Both biocatalysts (adsorbed or cross-linked) were stable at pH 10 and room temperature for 24 h (about 70% of retained activity). The cross-linked DmdNK preparation was used for the preparative synthesis of arabinosyladenine monophosphate (araA-MP) and fludarabine monophosphate (FaraAMP). Upon optimization of the reaction conditions (50 mM ammonium acetate, substrate/ATP ratio= 1:1.25, 2 mM MgCl2, 378C, pH 8) immobilized DmdNK afforded the title nucleotides with high conversion (>90%), whereas with the soluble enzyme lower conversions were achieved (78-87%). Arabinosyladenine monophosphate was isolated in 95% yield and high purity (96.5%).
- Serra, Immacolata,Conti, Silvia,Piskur, Jure,Clausen, Anders R.,Munch-Petersen, Birgitte,Terreni, Marco,Ubiali, Daniela
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p. 563 - 570
(2014/05/20)
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- The reaction of activated RNA species with aqueous fluoride ion: A convenient synthesis of nucleotide 5′-phosphorofluoridates and a note on the mechanism
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The chemistry of 5′-phosphorimidazolides of ribonucleosides is extended to include their reaction with alkali metal fluorides in aqueous solution. High yields of 5′-phosphorofluoridates are formed, especially with potassium fluoride, but no detectable oligomerization products were formed. A combination of HPLC, mass spectrometry, synthesis, kinetics, and NMR confirms the identities of the products. Judicious control of pH leads to higher yields in shorter reaction times. This new methodology contrasts favorably with other synthetic routes involving non-aqueous chemistry or aqueous chemistry with a nucleotide triphosphate.
- Aldersley, Michael F.,Joshi, Prakash C.,Schwartz, Herbert M.,Kirby, Anthony J.
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p. 1464 - 1466
(2014/03/21)
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- Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana
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Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized in mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2.7.1.21)-like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. The results obtained in the present study suggest a crucial role for the salvage of thymidine during early plant development. 2012 The Authors Journal compilation
- Clausen, Anders R.,Girandon, Lenart,Ali, Ashfaq,Knecht, Wolfgang,Rozpedowska, Elzbieta,Sandrini, Michael P. B.,Andreasson, Erik,Munch-Petersen, Birgitte,Piskur, Jure
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p. 3889 - 3897
(2013/01/13)
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- A single nuclease-resistant linkage in DNA as a versatile tool for the characterization of DNA lesions: Application to the guanine oxidative lesion g+34 generated by metalloporphyrin/KHSO5 reagent
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The oxidation of an oligonucleotide containing a single nuclease-resistant phosphodiester link, a stereoisomerically pure methylphosphonate, by manganese (Mn-TMPyP) or iron (Fe-TMPyP) porphyrin associated to KHSO5 allowed the isolation and characterization of a guanine lesion corresponding to an increase of mass of 34 amu as compared to guanine ( G+34 ), namely, 5-carboxamido-5-formamido-2-iminohydantoin. Enzymatic digestion of the damaged oligonucleotide afforded, apart from the undamaged nucleotide monomer pool, a unique dinucleotide doubly modified with a methylphosphonate and an oxidized guanine base that is suitable for NMR analysis. The method can be applied to the study of any DNA lesion. More importantly, the method can be extended to the analysis of DNA damage in a sequence context. Any preselected residue in a DNA sequence may be individually analyzed by the easy introduction of a single nuclease-resistant link at the 3′- or 5′-position.
- Tomaszewska, Agnieszka,Mourgues, Sophie,Guga, Piotr,Nawrot, Barbara,Pratviel, Genevieve
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p. 2505 - 2512
(2013/01/15)
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- Identification of critical ligand binding determinants in Mycobacterium tuberculosis adenosine-5′-phosphosulfate reductase
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Mycobacterium tuberculosis adenosine-5′-phosphosulfate (APS) reductase is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. To facilitate the development of potent and specific inhibitors of APS reductase, we have probed the molecular determinants that underlie binding and specificity through a series of substrate and product analogues. Our study highlights the importance of specific substitutent groups for substrate binding and provides functional evidence for ligand-specific conformational states. An active site model has been developed for M. tuberculosis APS reductase that is in accord with the results presented here as well as prior structural data reported for Pseudomonas aeruginosa APS reductase and related enzymes. This model illustrates the functional features required for the interaction of APS reductase with a ligand and provides a pharmacological roadmap for the rational design of small molecules as potential inhibitors of APS reductase present in human pathogens, including M. tuberculosis.
- Hong, Jiyoung A.,Bhave, Devayani P.,Carroll, Kate S.
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experimental part
p. 5485 - 5495
(2010/06/19)
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- Synthesis and stability of novel terminal phosphate-labeled nucleotides
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Novel compounds consisting of a nucleotide triphosphate labeled with a PEG linker and various terminal groups attached to the γ-phosphate of the nucleotide were constructed for use in efforts to produce a new class of DNA sequencer. The stability of these novel compounds was investigated to determine their utility as sequencing reagents. Hydrolysis rate constants were measured for both the natural nucleoside triphosphate dATP and novel dATP derivatives. The γ-labeled dATP was approximately 20-fold more stable to hydrolysis than dATP. Copyright Taylor & Francis Group, LLC.
- Reynolds, Bambi,Miller, Rachel,Williams, John G.,Anderson, Jon P.
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- An efficient reagent for the phosphorylation of deoxyribonucleosides, DNA oligonucleotides, and their thermolytic analogues
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(Chemical Equation Presented) The phosphoramidite 11 was prepared in three steps from methyl 2-mercaptoacetate and demonstrated efficiency in the synthesis of conventional 5′-/3′-phosphate/thiophosphate monoester derivatives of 2′-deoxyribonucleosides and DNA oligonucleotides. Moreover, the use of 11 has enabled the preparation of the dinucleoside phosphorothioate analogue 26 in high yields (>95%) with minimal cleavage (2%) of the thermolytic thiophosphate protecting group.
- Ausin, Cristina,Grajkowski, Andrzej,Cieslak, Jacek,Beaucage, Serge L.
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p. 4201 - 4204
(2007/10/03)
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- Synthesis of deoxyadenosine-5′-(35S)thiomonophosphate [dAMP(35S)] of high specific activity - A key intermediate for the synthesis of Sp-deoxyadenosine-5′-(α-35S)thiotriphosphate [Sp-dATP (α-35S)]
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Unprotected deoxyadenosine 1 was treated with an excess of phosphorus acid and stoichiometric proportions of N, N′-di-p- tolylcarbodiimide in anhydrous pyridine to give deoxyadenosine-5′-monophosphite 2. The latter wax activated with trimethylsilyl chloride followed by sulphurisation with elemental 35S (specific activity > 1000 Ci/mmol) in toluene solution to give deoxyadenosine5′-(35S)-thiomonophosphate [dAMP(35S)] 3. Enzymatic conversion of deoxyadenosine-5′-(35S)-thiomonophosphate to Sp-deoxyadenosine-5′-(α-35S)-thiotriphosphate [Sp-dATP (α-35S)] 5 was carried out following a standard reaction protocol. Copyright
- Mathew,Ravi,Sontakke,Chander
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- Enzymatic phosphorylation of unnatural nucleosides
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In an effort to expand the genetic alphabet, a number of unnatural, predominantly hydrophobic, nucleoside analogues have been developed which pair selectively in duplex DNA and during enzymatic synthesis. Significant progress has been made toward the efficient in vitro replication of DNA containing these base pairs. However, the in vivo expansion of the genetic alphabet will require that the unnatural nucleoside triphosphates be available within the cell at sufficient concentrations for DNA replication. We report our initial efforts toward the development of an unnatural in vivo nucleoside phosphorylation pathway that is based on nucleoside salvage enzymes. The first step of this pathway is catalyzed by the D. melanogaster nucleoside kinase, which catalyzes the phosphorylation of nucleosides to the corresponding monophosphates. We demonstrate that each unnatural nucleoside is phosphorylated with a rate that should be sufficient for the in vivo replication of DNA.
- Wu, Yiqin,Fa, Ming,Tae, Eunju Lee,Schultz, Peter G.,Romesberg, Floyd E.
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p. 14626 - 14630
(2007/10/03)
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- A new method for the phosphorylation of nucleosides
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A new phosphorylating reagent, 2-O-(4,4'-dimethoxytrityl)ethylsulfonylethan-2'-yl-phosphate (1), was developed for the phosphorylation of primary and secondary alcohols of nucleosides. In the many examples studied, yields in the phosphorylation step were excellent (~80 to 95%). There is potential for wide applicability of this procedure, not only in nucleoside and nucleotide chemistry, but also in the phosphorylation of biomolecules such as carbohydrates and amino acids. (C) 2000 Elsevier Science Ltd.
- Taktakishvili,Nair
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p. 7173 - 7176
(2007/10/03)
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- Differences in kinetic properties of pure recombinant human and mouse deoxycytidine kinase
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Human and mouse deoxycytidine kinase (dCK) (EC 2.7.1.74) were cloned and expressed in Escherichia coli. Michaelis-Menten kinetics were determined for the purified enzymes with 2'-deoxycytidine (dCyd), 2'-deoxyadenosine (dAdo), 2-chloro-2'-deoxyadenosine (CdA), 2',3'dideoxycytidine (ddCyd) and 9-β-D-arabinofuranosylguanine (araG) as substrates and ATP and UTP as phosphate donors. Both human and mouse dCK showed highest affinity to dCvd with K(m) values of 0.05-0.2 μM. The anti-leukaemic compound CdA was the superior substrate of the nucleoside analogues tested. Both enzymes were able to efficiently utilize ATP and UTP as phosphate donors. However, the use of UTP instead of ATP as phosphate donor decreased K(m) values for all substrates investigated. The kinetic properties of mouse and human dCK differed in that the human enzyme showed higher affinity for the substrates dAdo, CdA, ddCyd and araG. The human enzyme also showed higher affinity for ATP and UTP. The ability to phosphorylate dCyd was, however, similar for both human and mouse dCK. At physiological concentration of the feedback inhibitor dCTP, mouse dCK showed lower activity than human dCK for all substrates investigated.
- Johansson, Magnus,Karlsson, Anna
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p. 163 - 168
(2007/10/03)
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- TRITYLOXYETHYLAMINO GROUP FOR THE PROTECTION OF PHOSPHORYL GROUP IN OLIGONUCLEOTIDE SYNTHESIS
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Trityloxyethylamino group was used for the protection of 5'-phosphoryl group of deoxyribonucleoside.This group is not only protecting group of phosphate also the intermediate for the synthesis of d-ppA and d-pppA.
- Tanaka, Toshiki,Yamada, Yasuki,Ikehara, Morio
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p. 5641 - 5644
(2007/10/02)
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- Chemical Synthesis of 5'-Phosphorylated DNA Fragments and Their Constituents
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Phosphoryl tris-triazole has been applied to the synthesis of DNA fragments and their constituents bearing 5'-phosphomonoester function and to the preparation of deoxynucleoside 5'- or 3'-diphosphates.
- Jankowska, Jadwiga,Stawinski, Jacek
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- SYNTHESIS OF 5'-PHOSPHORYLATED DNA FRAGMENTS
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Morpholino phosphorobis-3-nitro-1,2,4-triazolidate (1) has been applied to the phosphorylation of partially protected (5'-OH free) DNA intermediates (2).Using this procedure, the following types of compounds were synthesized: pBp(Bp)nB; AppBp(Bp)nB and ppBp(Bp)nB.
- Marel, G. van der,Veeneman, G.,Boom, J. H. van
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p. 1463 - 1466
(2007/10/02)
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